Multiplex organoid mutagenesis reveals gene combinations driving transformation, pathology and chemosensitivity in ovarian carcinoma

High-grade serous ovarian carcinoma (HGSC) is the sixth leading cause of cancer-related death among women. Many cases arise from the Fallopian tubal epithelium (TE), exhibit numerous mutations, and present heterogenous pathological features. However, the contribution of specific mutation combinations to cellular transformation, pathological phenotype and chemotherapeutic response remains poorly understood. Here, we used a Trp53-deficient mouse TE-derived organoid platform to perform combinatorial CRISPR mutagenesis of 20 candidate HGSC driver genes. Mutations in Nf1, Cdkn2a and Map2k4 were most frequently observed in transformed organoids. Upon transplantation into mice, those containing Map2k4 mutations predominantly gave rise to papillary-glandular histology, whereas those containing Nf1 mutations formed more mesenchymal-like carcinomas. Transcriptomic analysis revealed that tumors bearing Nf1 mutations overexpressed the long non-coding RNA Pvt1, a marker associated with poor prognosis in HGSC patients, regardless of pathological phenotype Map2k4-mutant organoids were more sensitive to paclitaxel and niraparib, while Nf1-mutant combinations responded better to trametinib. Notably, the removal of Rho kinase inhibitor (ROCKi) reduced trametinib sensitivity in both Map2k4- and Nf1-mutant organoids, underscoring the importance of culture conditions and potential antagonistic drug interactions in organoid-based drug screens. Collectively, our results demonstrate that TE-derived organoids coupled with combinatorial CRISPR mutagenesis provide a powerful system to unravel the genetic and phenotypic complexity of HGSC. In particular, we found that Map2k4 functions as a tumor suppressor that shapes distinct tumor histology and chemosensitivity, suggesting it as a potential therapeutic target in select HGSC cases.